All living organisms require a continuous supply of usable energy to maintain their structure and to grow. In aerobic chemoheterotrophs, a variety of substrates can be oxidized through the action of specific dehydrogenases. In eukaryotes, many of these enzymes are found in the mitochondria; in prokaryotes, they are often localized in the plasma membrane. Since most aerobic chemoheterotrophs can utilize a number of oxidizable substrates, choices must be made among them. While the use of alternative substrates may be determined by the rates of formation of the individual enzymes or their kinetic properties, it may also be determined by the membrane association process. Of the enzymatic systems that might be used to explore the issues of protein localization and global regulation, the catabolism of L- proline is of particular interest because this amino acid serves at least three different metabolic functions. Although it is one of the 20 amino acids from which proteins are formed, proline can also be degraded as a carbon, nitrogen, or energy source, or stored in the cytoplasm during conditions of osmotic stress. Defects in proline catabolism underlie one form of hyperprolinemia and can prevent its use as a carbon and energy source. The goal of this project is to initiate a genetic study of the role of membrane association in the regulation of L-proline dehydrogenase in Escherichia coli. The following experiments will be performed: 1) proline dehydrogenase activities will be measured in a series of E. coli strains containing known mutations in genes involved in protein export and oxidative metabolism; 2) the effects of alternative substrates on the levels of proline dehydrogenase in wild-type strains will be determined; and 3) a set of new mutants that exhibit increased proline dehydrogenase activity in the presence of alternative substrates will be isolated and characterized. These studies should provide a better understanding of the genetic basis of protein localization and new insights into the mechanisms of global metabolic control.